Department of Cardiology, Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, PR China.
Department of Geriatric Intensive Medicine, Guangdong Provincial Geriatrics Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, PR China.
Int Immunopharmacol. 2023 May;118:110105. doi: 10.1016/j.intimp.2023.110105. Epub 2023 Apr 3.
Contrast-induced acute kidney injury (CI-AKI) is a common complication following percutaneous coronary intervention in coronary artery disease (CAD) patients with >30% incidence. Klotho is a multifunctional protein that inhibits oxidative stress and inflammation, but its role in CI-AKI is poorly understood. The present study aimed to explore the effects of klotho in CI-AKI.
Six-week-old mice and HK-2 were divided into the control, contrast medium (CM), CM + klotho, and klotho groups. H&E staining evaluated kidney injury. Scr and BUN showed renal function. DHE probe and ELISA kit detected the levels of reactive oxygen species (ROS) in kidney tissue, superoxide dismutase (SOD), and malondialdehyde (MDA) in serum. Western blot detected the expressions of NF-κB and phosphorylated NF-κB (p-NF-κB) and pyroptosis-related protein levels of NLRP3, caspase-1, GSDMD, and cleaved-GSDMD in the kidney of CI-AKI mice. CCK-8 and lactate dehydrogenase (LDH) activity assays determined cell viability and damage. Fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) and enzyme-linked immunosorbent assay (ELISA) tested oxidative stress-related indicators. These included intracellular reactive oxygen species (ROS), superoxidase dismutase (SOD), and malondialdehyde (MDA). IL-6, TNF-α, IL-1β, and IL-18 in the cell supernatant were tested by ELISA assay and used to reflect inflammation responses. Propidium iodide (PI) staining showed the cell death of HK-2. The expressions of NF-κB, p-NF-κB and pyroptosis-related protein levels of NLRP3, caspase-1, GSDMD, and cleaved-GSDMD were detected by Western blot.
Exogenous klotho administration reduced kidney histopathological alterations and improved renal function in vivo. The levels of reactive oxygen species (ROS) in renal tissue, superoxide dismutase (SOD), and malondialdehyde (MDA) in serum decreased after the klotho intervention. The expression levels of p-NF-κB and pyroptosis-related proteins, including NLRP3, caspase-1, GSDMD, and cleaved-GSDMD, were decreased in CI-AKI mice after the klotho intervention. In vitro, klotho significantly inhibited CM-induced oxidative stress and the production of IL-6 and TNF-α. Moreover, it was found that klotho inhibited the activation of p-NF-κB and down-regulated pyroptosis-related protein (NLRP3, caspase-1, GSDMD, and cleaved-GSDMD).
Klotho has a protective effect on CI-AKI via suppressing oxidative stress, inflammation, and NF-κB/NLRP3-mediated pyroptosis that contributes to the potential therapy of CI-AKI.
对比剂诱导的急性肾损伤（CI-AKI）是冠心病（CAD）患者经皮冠状动脉介入治疗后常见的并发症，发生率超过 30%。Klotho 是一种多功能蛋白，可抑制氧化应激和炎症，但它在 CI-AKI 中的作用尚不清楚。本研究旨在探讨 Klotho 在 CI-AKI 中的作用。
将 6 周龄小鼠和 HK-2 细胞分为对照组、对比剂（CM）组、CM+Klotho 组和 Klotho 组。H&E 染色评估肾脏损伤。血清肌酐（Scr）和血尿素氮（BUN）反映肾功能。二氢乙二肟（DHE）探针和 ELISA 试剂盒检测肾组织中活性氧（ROS）、超氧化物歧化酶（SOD）和丙二醛（MDA）的水平。Western blot 检测 NF-κB 和磷酸化 NF-κB（p-NF-κB）以及 Klotho 处理后 CI-AKI 小鼠肾脏中 NLRP3、半胱氨酸天冬氨酸蛋白酶-1（caspase-1）、Gasdermin D（GSDMD）和 cleaved-GSDMD 等焦亡相关蛋白的表达。CCK-8 和乳酸脱氢酶（LDH）活性测定评估细胞活力和损伤。荧光探针二氯二氢荧光素二乙酸酯（DCFH-DA）和酶联免疫吸附测定（ELISA）检测细胞内活性氧（ROS）、超氧化物歧化酶（SOD）和丙二醛（MDA）等氧化应激相关指标。ELISA 检测细胞上清液中白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18），反映炎症反应。碘化丙啶（PI）染色显示 HK-2 细胞死亡。Western blot 检测 NF-κB、p-NF-κB 和焦亡相关蛋白 NLRP3、caspase-1、GSDMD 和 cleaved-GSDMD 的表达。
外源性 Klotho 给药可减轻体内肾脏组织学改变并改善肾功能。Klotho 干预后，肾组织中活性氧（ROS）水平以及血清中超氧化物歧化酶（SOD）和丙二醛（MDA）水平降低。Klotho 干预后，CI-AKI 小鼠 p-NF-κB 和焦亡相关蛋白（NLRP3、caspase-1、GSDMD 和 cleaved-GSDMD）的表达水平降低。体外实验表明，Klotho 可显著抑制 CM 诱导的氧化应激和白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的产生。此外，发现 Klotho 抑制 p-NF-κB 的激活并下调焦亡相关蛋白（NLRP3、caspase-1、GSDMD 和 cleaved-GSDMD）。
Klotho 通过抑制氧化应激、炎症和 NF-κB/NLRP3 介导的焦亡对 CI-AKI 具有保护作用，为 CI-AKI 的潜在治疗提供了依据。
